Transgenic plants with enhanced agronomic traits

ABSTRACT

This invention provides transgenic plant cells with recombinant DNA for expression of proteins that are useful for imparting enhanced agronomic trait(s) to transgenic crop plants. This invention also provides transgenic plants and progeny seed comprising the transgenic plant cells where the plants are selected for having an enhanced trait selected from the group of traits consisting of enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil. Also disclosed are methods for manufacturing transgenic seed and plants with enhanced traits.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 USC § 119(e) of U.S. provisional application Ser. No. 61/013,179, filed Dec. 12, 2007, incorporated herein by reference in its entirety.

INCORPORATION OF SEQUENCE LISTING

Two copies of the sequence listing (Copy 1 and Copy 2) and a computer readable form (CRF) of the sequence listing, all on CD-Rs, each containing the text file named “38-21-55579-B_seqListing.txt”, which is 5,484,544 bytes (measured in MS-WINDOWS), were created on Dec. 8, 2008 and are incorporated herein by reference.

INCORPORATION OF LARGE TABLE

Two copies of a large table (Copy 1 and Copy 2) containing a folder “pfamdir” on CD-Rs are incorporated herein by reference in their entirety. Folder “pfamdir” contains 10 Pfam Hidden Markov Models. The CD-Rs were created on Dec. 8, 2008, having a total size of 864,256 bytes (measured in MS-WINDOWS).

FIELD OF THE INVENTION

Disclosed herein are recombinant DNA useful for providing enhanced traits to transgenic plants, seeds, pollen, plant cells and plant nuclei of such transgenic plants, methods of making and using such recombinant DNA, plants, seeds, pollen, plant cells and plant nuclei. Also disclosed are methods of producing hybrid corn seed comprising such recombinant DNA.

SUMMARY OF THE INVENTION

An aspect of this invention provides recombinant DNA constructs comprising polynucleotides characterized by an encoded protein having amino acids representing a protein family domain module as described in Table 10. Another aspect of this invention provides recombinant DNA constructs comprising polynucleotides characterized by an encoded protein with an amino acid sequence that is at least 90% identical to a corresponding consensus sequence defined in table 8. Yet another aspect of this invention provides recombinant DNA constructs comprising polynucleotides characterized by reference to SEQ ID NO:1-10 and the cognate proteins with amino acid sequences having reference to SEQ ID NO:11-20. The recombinant DNA constructs are useful for providing enhanced traits when stably integrated into the chromosomes and expressed in the nuclei of transgenic plants cells. In most aspects of the invention the recombinant DNA constructs, when expressed in a plant cell, provide for expression of cognate proteins. In particular aspects of the invention the recombinant DNA constructs for expressing cognate proteins are characterized by cognate amino acid sequence that have at least 95% identity over at least 95% of the length of a reference sequence in the group of SEQ ID NOs: 12-16, and 20 when the amino acid sequence is aligned to the reference sequence. In particularly specific embodiments of the invention, the recombinant DNA constructs comprise polynucleotide stacks characterized by cognate proteins having amino acid sequences that have at least 95% identity over at least 95% of the length of reference sequences 13-14 when the amino acid sequences are aligned to the reference sequences. In some aspects of the invention, i.e. the recombinant DNA constructs are characterized as being constructed with sense-oriented and anti-sense-oriented polynucleotides from SEQ ID NOs: 1, and 7-9 which, when expressed in a plant cell, provide for the suppression of cognate proteins having amino acid sequences that have at least 95% identity over at least 95% of the length of a reference sequence in the group consisting of SEQ ID NOs: 11 and 17-19.

In practical aspects of this invention the recombinant DNA constructs of the invention are stably integrated into the chromosome of a plant cell nucleus.

This invention also provides transgenic plant cells comprising the stably integrated recombinant DNA constructs of the invention, transgenic plants and seeds comprising a plurality of such transgenic plant cells and transgenic pollen of such plants. Such transgenic plants are selected from a population of transgenic plants regenerated from plant cells transformed with recombinant DNA constructs by screening transgenic plants for an enhanced trait as compared to control plants. The enhanced trait is one or more of enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil.

In another aspect of the invention the plant cells, plants, seeds, and pollen further comprise DNA expressing a protein that provides tolerance from exposure to an herbicide applied at levels that are lethal to a wild type plant cell.

This invention also provides methods for manufacturing non-natural, transgenic seed that can be used to produce a crop of transgenic plants with an enhanced trait resulting from expression of a stably-integrated recombinant DNA construct. More specifically, the method comprises (a) screening a population of plants for an enhanced trait and a recombinant DNA construct, where individual plants in the population can exhibit the trait at a level less than, essentially the same as or greater than the level that the trait is exhibited in control plants, (b) selecting from the population one or more plants that exhibit the trait at a level greater than the level that said trait is exhibited in control plants, (c) collecting seed from a selected plant, (d) verifying that the recombinant DNA is stably integrated in said selected plants, (e) analyzing tissue of a selected plant to determine the production or suppression of a protein having the function of a protein encoded by nucleotides in a sequence of one of SEQ ID NOs:1-10. In one aspect of the invention, the plants in the population further comprise DNA expressing a protein that provides tolerance to exposure to a herbicide applied at levels that are lethal to wild type plant cells and the selecting is affected by treating the population with the herbicide, e.g. a glyphosate, dicamba, or glufosinate compound. In another aspect of the invention the plants are selected by identifying plants with the enhanced trait. The methods are especially useful for manufacturing corn, soybean, cotton, canola, alfalfa, wheat, rice, sugarcane or sugar beet seed.

Another aspect of the invention provides a method of producing hybrid corn seed comprising acquiring hybrid corn seed from a herbicide tolerant corn plant which also has stably-integrated, recombinant DNA construct comprising a promoter that is (a) functional in plant cells and (b) is operably linked to DNA that encodes or suppresses a protein having the function of a protein encoded by nucleotides in a sequence of one of SEQ ID NOs:1-10. The methods further comprise producing corn plants from said hybrid corn seed, wherein a fraction of the plants produced from said hybrid corn seed is homozygous for said recombinant DNA, a fraction of the plants produced from said hybrid corn seed is hemizygous for said recombinant DNA, and a fraction of the plants produced from said hybrid corn seed has none of said recombinant DNA; selecting corn plants which are homozygous and hemizygous for said recombinant DNA by treating with an herbicide; collecting seed from herbicide-treated-surviving corn plants and planting said seed to produce further progeny corn plants; repeating the selecting and collecting steps at least once to produce an inbred corn line; and crossing the inbred corn line with a second corn line to produce hybrid seed.

Another aspect of the invention provides a method of selecting a plant comprising plant cells of the invention by using an immunoreactive antibody to detect the presence or absence of protein expressed or suppressed by recombinant DNA in seed or plant tissue. Yet another aspect of the invention provides anti-counterfeit milled seed having, as an indication of origin, plant cells of this invention.

Still other aspects of this invention relate to transgenic plants with enhanced water use efficiency or enhanced nitrogen use efficiency. For instance, this invention provides methods of growing a corn, cotton, soybean, or canola crop without irrigation water comprising planting seed having plant cells of the invention which are selected for enhanced water use efficiency. Alternatively methods comprise applying reduced irrigation water, e.g. providing up to 300 millimeters of ground water during the production of a corn crop. This invention also provides methods of growing a corn, cotton, soybean or canola crop without added nitrogen fertilizer comprising planting seed having plant cells of the invention which are selected for enhanced nitrogen use efficiency.

Another aspect of the invention provides a mixture comprising plants cells and an antibody to a protein produced in the cells where the protein has an amino acid sequence that has at least 95% identity over at least 95% of the length of a reference sequence selected from the group consisting of SEQ ID NO: 11-20 when the sequence is aligned to the reference sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-4 are plasmid maps.

DETAILED DESCRIPTION OF THE INVENTION

In the attached sequence listing:

SEQ ID NO:1-10 are nucleotide sequences of the coding strand of DNA for “genes” used in the recombinant DNA imparting an enhanced trait in plant cells, i.e. each represents a coding sequence for a protein;

SEQ ID NO: 11-20 are amino acid sequences of the cognate protein of the “genes” with nucleotide coding sequences 1-10;

SEQ ID NO: 21-1351 are amino acid sequences of homologous proteins;

SEQ ID NO: 1352 is a nucleotide sequence of a base plasmid vector useful for corn transformation;

SEQ ID NO: 1353 is a nucleotide sequence of a base plasmid vector useful for soybean and canola transformation;

SEQ ID NO: 1354 is a nucleotide sequence of a base plasmid vector useful for cotton transformation;

SEQ ID NO: 1355 is a nucleotide sequence of a base plasmid vector useful for co-transformation to produce gene stacks in corn;

SEQ ID NO: 1356-1358 are consensus sequences.

Table 8 lists the protein SEQ ID NOs and their corresponding consensus SEQ ID NOs.

As used herein a “plant cell” means a plant cell that is transformed with stably-integrated, non-natural, recombinant DNA, e.g. by Agrobacterium-mediated transformation or by bombardment using microparticles coated with recombinant DNA or other means. A plant cell of this invention can be an originally-transformed plant cell that exists as a microorganism or as a progeny plant cell that is regenerated into differentiated tissue, e.g. into a transgenic plant with stably-integrated, non-natural recombinant DNA, or seed or pollen derived from a progeny transgenic plant.

As used herein a “transgenic plant” means a plant whose genome has been altered by the stable integration of recombinant DNA. A transgenic plant includes a plant regenerated from an originally-transformed plant cell and progeny transgenic plants from later generations or crosses of a transformed plant.

As used herein “recombinant DNA” means DNA which has been a genetically engineered and constructed outside of a cell including DNA containing naturally occurring DNA or cDNA or synthetic DNA.

As used herein “consensus sequence” means an artificial sequence of amino acids in a conserved region of an alignment of amino acid sequences of homologous proteins, e.g. as determined by a CLUSTALW alignment of amino acid sequence of homolog proteins.

As used herein a “homolog” means a protein in a group of proteins that perform the same biological function, e.g. proteins that belong to the same Pfam protein family and that provide a common enhanced trait in transgenic plants of this invention. Homologs are expressed by homologous genes. With reference to homologous genes, homologs include orthologs, i.e. genes expressed in different species that evolved from a common ancestral genes by speciation and encode proteins retain the same function, but do not include paralogs, i.e. genes that are related by duplication but have evolved to encode proteins with different functions. Homologous genes include naturally occurring alleles and artificially-created variants. Degeneracy of the genetic code provides the possibility to substitute at least one base of the protein encoding sequence of a gene with a different base without causing the amino acid sequence of the polypeptide produced from the gene to be changed. When optimally aligned, homolog proteins have at least 60% identity, more preferably about 65% or higher, more preferably about 70% or higher, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, and even more preferably at least 90% identity over the full length of a protein identified as being associated with imparting an enhanced trait when expressed in plant cells. In one aspect of the invention homolog proteins have an amino acid sequence that has at least 90% identity to a consensus amino acid sequence of proteins and homologs disclosed herein.

Homologs are identified by comparison of amino acid sequence, e.g. manually or by use of a computer-based tool using known homology-based search algorithms such as those commonly known and referred to as BLAST, FASTA, and Smith-Waterman. A local sequence alignment program, e.g. BLAST, can be used to search a database of sequences to find similar sequences, and the summary Expectation value (E-value) used to measure the sequence base similarity. Because a protein hit with the best E-value for a particular organism may not necessarily be an ortholog, i.e. have the same function, or be the only ortholog, a reciprocal query is used to filter hit sequences with significant E-values for ortholog identification. The reciprocal query entails search of the significant hits against a database of amino acid sequences from the base organism that are similar to the sequence of the query protein. A hit can be identified as an ortholog, when the reciprocal query's best hit is the query protein itself or a protein encoded by a duplicated gene after speciation. A further aspect of the homologs encoded by DNA useful in the transgenic plants of the invention are those proteins that differ from a disclosed protein as the result of deletion or insertion of one or more amino acids in a native sequence.

“Percent identity” describes the extent to which the sequences of DNA or protein segments are invariant throughout a window of alignment of sequences, for example nucleotide sequences or amino acid sequences. An “identity fraction” for a sequence aligned with a reference sequence is the number of identical components which are shared by the sequences, divided by the length of the alignment not including gaps introduced by the alignment algorithm. “Percent identity” (“% identity”) is the identity fraction times 100. Percent identity is calculated over the aligned length preferably using a local alignment algorithm, such as BLASTp. As used herein, sequences are “aligned” when the alignment produced by BLASTp has a minimal e-value.

“Pfam” is a large collection of multiple sequence alignments and hidden Markov models covering many common protein families, e.g. Pfam version 19.0 (December 2005) contains alignments and models for 8183 protein families and is based on the Swissprot 47.0 and SP-TrEMBL 30.0 protein sequence databases. See S. R. Eddy, “Profile Hidden Markov Models”, Bioinformatics 14:755-763, 1998. The Pfam database is currently maintained and updated by the Pfam Consortium. The alignments represent some evolutionary conserved structure that has implications for the protein's function. Profile hidden Markov models (profile HMMs) built from the protein family alignments are useful for automatically recognizing that a new protein belongs to an existing protein family even if the homology by alignment appears to be low.

Protein domains are identified by querying the amino acid sequence of a protein against Hidden Markov Models which characterize protein family domains (“Pfam domains”) using HMMER software, which is available from the Pfam Consortium. The HMMER software is also disclosed in patent application publication US 2008/0148432 A1 incorporated herein by reference. A protein domain meeting the gathering cutoff for the alignment of a particular Pfam domain is considered to contain the Pfam domain.

A “Pfam domain module” is a representation of Pfam domains in a protein, in order from N terminus to C terminus. In a Pfam domain module individual Pfam domains are separated by double colons “::”. The order and copy number of the Pfam domains from N to C terminus are attributes of a Pfam domain module. Although the copy number of repetitive domains is important, varying copy number often enables a similar function. Thus, a Pfam domain module with multiple copies of a domain should define an equivalent Pfam domain module with variance in the number of multiple copies. A Pfam domain module is not specific for distance between adjacent domains, but contemplates natural distances and variations in distance that provide equivalent function. The Pfam database contains both narrowly- and broadly-defined domains, leading to identification of overlapping domains on some proteins. A Pfam domain module is characterized by non-overlapping domains. Where there is overlap, the domain having a function that is more closely associated with the function of the protein (based on the E value of the Pfam match) is selected.

Once one DNA is identified as encoding a protein which imparts an enhanced trait when expressed in transgenic plants, other DNA encoding proteins with the same Pfam domain module are identified by querying the amino acid sequence of protein encoded by candidate DNA against the Hidden Markov Models which characterizes the Pfam domains using HMMER software. Candidate proteins meeting the same Pfam domain module are in the protein family and have cognate DNA that is useful in constructing recombinant DNA for the use in the plant cells of this invention. Hidden Markov Model databases for use with HMMER software in identifying DNA expressing protein with a common Pfam domain module for recombinant DNA in the plant cells of this invention are included in the large table incorporated into this application.

The HMMER software and Pfam databases (version 19.0) were used to identify known domains in the proteins corresponding to amino acid sequence of SEQ ID NO: 11 through SEQ ID NO: 16 and SEQ ID NO: 20. All DNA encoding proteins that have scores higher than the gathering cutoff disclosed in Table 11 by Pfam analysis disclosed herein can be used in recombinant DNA of the plant cells of this invention, e.g. for selecting transgenic plants having enhanced agronomic traits. The relevant Pfams modules for use in this invention, as more specifically disclosed below, are Cu-oxidase_(—)3::Cu-oxidase::Cu-oxidase_(—)2, Flavodoxin_(—)1, Glyco_transf_(—)20::Trehalose_PPase, Aminotran_(—)1_(—)2, and B3::Auxin_resp::AUX_IAA, for which the databases are included in the appended computer listing.

As used herein “promoter” means regulatory DNA for initializing transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells whether or not its origin is a plant cell, e.g. is it well known that Agrobacterium promoters are functional in plant cells. Thus, plant promoters include promoter DNA obtained from plants, plant viruses and bacteria such as Agrobacterium and Bradyrhizobium bacteria. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, or seeds. Such promoters are referred to as “tissue preferred”. Promoters that initiate transcription only in certain tissues are referred to as “tissue specific”. A “cell type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An “inducible” or “repressible” promoter is a promoter which is under environmental control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions, or certain chemicals, or the presence of light. Tissue specific, tissue preferred, cell type specific, and inducible promoters constitute the class of “non-constitutive” promoters. A “constitutive” promoter is a promoter which is active under most conditions.

As used herein “operably linked” means the association of two or more DNA fragments in a recombinant DNA construct so that the function of one, e.g. protein-encoding DNA, is controlled by the other, e.g. a promoter.

As used herein “expressed” means produced, e.g. a protein is expressed in a plant cell when its cognate DNA is transcribed to mRNA that is translated to the protein.

As used herein “suppressed” means decreased, e.g. a protein is suppressed in a plant cell when there is a decrease in the amount and/or activity of the protein in the plant cell. The presence or activity of the protein can be decreased by any amount up to and including a total loss of protein expression and/or activity.

As used herein a “control plant” means a plant that does not contain the recombinant DNA that imparts an enhanced trait. A control plant is used to identify and select a transgenic plant that has an enhanced trait. A suitable control plant can be a non-transgenic plant of the parental line used to generate a transgenic plant, i.e. devoid of recombinant DNA. A suitable control plant may in some cases be a progeny of a hemizygous transgenic plant line that does not contain the recombinant DNA, known as a negative segregant.

As used herein an “enhanced trait” means a characteristic of a transgenic plant that includes, but is not limited to, an enhance agronomic trait characterized by enhanced plant morphology, physiology, growth and development, yield, nutritional enhancement, disease or pest resistance, or environmental or chemical tolerance. In more specific aspects of this invention enhanced trait is selected from group of enhanced traits consisting of enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil. In an important aspect of the invention the enhanced trait is enhanced yield including increased yield under non-stress conditions and increased yield under environmental stress conditions. Stress conditions may include, for example, drought, shade, fungal disease, viral disease, bacterial disease, insect infestation, nematode infestation, cold temperature exposure, heat exposure, osmotic stress, reduced nitrogen nutrient availability, reduced phosphorus nutrient availability and high plant density. “Yield” can be affected by many properties including without limitation, plant height, pod number, pod position on the plant, number of internodes, incidence of pod shatter, grain size, efficiency of nodulation and nitrogen fixation, efficiency of nutrient assimilation, resistance to biotic and abiotic stress, carbon assimilation, plant architecture, resistance to lodging, percent seed germination, seedling vigor, and juvenile traits. Yield can also be affected by efficiency of germination (including germination in stressed conditions), growth rate (including growth rate in stressed conditions), ear number, seed number per ear, seed size, composition of seed (starch, oil, protein) and characteristics of seed fill.

Increased yield of a transgenic plant of the present invention can be measured in a number of ways, including test weight, seed number per plant, seed weight, seed number per unit area (i.e. seeds, or weight of seeds, per acre), bushels per acre, tons per acre, or kilo per hectare. For example, corn yield may be measured as production of shelled corn kernels per unit of production area, for example in bushels per acre or metric tons per hectare, often reported on a moisture adjusted basis, for example at 15.5 percent moisture. Increased yield may result from improved utilization of key biochemical compounds, such as nitrogen, phosphorous and carbohydrate, or from improved responses to environmental stresses, such as cold, heat, drought, salt, and attack by pests or pathogens. Recombinant DNA used in this invention can also be used to provide plants having improved growth and development, and ultimately increased yield, as the result of modified expression of plant growth regulators or modification of cell cycle or photosynthesis pathways. Also of interest is the generation of transgenic plants that demonstrate enhanced yield with respect to a seed component that may or may not correspond to an increase in overall plant yield. Such properties include enhancements in seed oil, seed molecules such as protein and starch, oil components as may be manifest by an alterations in the ratios of seed components.

Recombinant DNA constructs are assembled using methods well known to persons of ordinary skill in the art and typically comprise a promoter operably linked to DNA, the expression of which provides the enhanced agronomic trait. Other construct components may include additional regulatory elements, such as 5′ leaders and introns for enhancing transcription, 3′ untranslated regions (such as polyadenylation signals and sites), DNA for transit or signal peptides.

Numerous promoters that are active in plant cells have been described in the literature. These include promoters present in plant genomes as well as promoters from other sources, including nopaline synthase (NOS) promoter and octopine synthase (OCS) promoters carried on tumor-inducing plasmids of Agrobacterium tumefaciens and the CaMV35S promoters from the cauliflower mosaic virus as disclosed in U.S. Pat. Nos. 5,164,316 and 5,322,938. Useful promoters derived from plant genes are found in U.S. Pat. No. 5,641,876 which discloses a rice actin promoter, U.S. Pat. No. 7,151,204 which discloses a maize chloroplast aldolase promoter and a maize aldolase (FDA) promoter, and US Patent Application Publication 2003/0131377 A1 which discloses a maize nicotianamine synthase promoter. These and numerous other promoters that function in plant cells are known to those skilled in the art and available for use in recombinant polynucleotides of the present invention to provide for expression of desired genes in transgenic plant cells.

Furthermore, the promoters may be altered to contain multiple “enhancer sequences” to assist in elevating gene expression. Such enhancers are known in the art. By including an enhancer sequence with such constructs, the expression of the selected protein may be enhanced. These enhancers often are found 5′ to the start of transcription in a promoter that functions in eukaryotic cells, but can often be inserted upstream (5′) or downstream (3′) to the coding sequence. In some instances, these 5′ enhancing elements are introns. Particularly useful as enhancers are the 5′ introns of the rice actin 1 (see U.S. Pat. No. 5,641,876) and rice actin 2 genes, the maize alcohol dehydrogenase gene intron, the maize heat shock protein 70 gene intron (U.S. Pat. No. 5,593,874) and the maize shrunken 1 gene. See also US Patent Application Publication 2002/0192813A1 which discloses 5′, 3′ and intron elements useful in the design of effective plant expression vectors.

In other aspects of the invention, sufficient expression in plant seed tissues is desired to affect improvements in seed composition. Exemplary promoters for use for seed composition modification include promoters from seed genes such as napin as disclosed in U.S. Pat. No. 5,420,034, maize L3 oleosin as disclosed in U.S. Pat. No. 6,433,252), zein Z27 as disclosed by Russell et al. (1997) Transgenic Res. 6 (2):157-166), globulin 1 as disclosed by Belanger et al (1991) Genetics 129:863-872), glutelin 1 as disclosed by Russell (1997) supra), and peroxiredoxin antioxidant (Per1) as disclosed by Stacy et al. (1996) Plant Mol. Biol. 31 (6): 1205-1216.

Recombinant DNA constructs useful in this invention will also generally include a 3′ element that typically contains a polyadenylation signal and site. Well-known 3′ elements include those from Agrobacterium tumefaciens genes such as nos 3′, tml 3′, tmr 3′, tms 3′, ocs 3′, tr7 3′, for example disclosed in U.S. Pat. No. 6,090,627; 3′ elements from plant genes such as wheat (Triticum aesevitum) heat shock protein 17 (Hsp17 3′), a wheat ubiquitin gene, a wheat fructose-1,6-biphosphatase gene, a rice glutelin gene, a rice lactate dehydrogenase gene and a rice beta-tubulin gene, all of which are disclosed in US Patent Application Publication 2002/0192813 A1; and the pea (Pisum sativum) ribulose biphosphate carboxylase gene (rbs 3′), and 3′ elements from the genes within the host plant.

Constructs and vectors may also include a transit peptide for targeting of a gene to a plant organelle, particularly to a chloroplast, leucoplast or other plastid organelle. For descriptions of the use of chloroplast transit peptides see U.S. Pat. No. 5,188,642 and U.S. Pat. No. 5,728,925. For description of the transit peptide region of an Arabidopsis EPSPS gene useful in the present invention, see Klee, H. J. et al (MGG (1987) 210:437-442).

Recombinant DNA constructs for gene suppression can be designed for any of a number the well-known methods for suppressing transcription of a gene, the accumulation of the mRNA corresponding to that gene or preventing translation of the transcript into protein. Posttranscriptional gene suppression can be practically effected by transcription of RNA that forms double-stranded RNA (dsRNA) having homology to mRNA produced from a gene targeted for suppression.

Gene suppression can also be achieved by insertion mutations created by transposable elements may also prevent gene function. For example, in many dicot plants, transformation with the T-DNA of Agrobacterium may be readily achieved and large numbers of transformants can be rapidly obtained. Also, some species have lines with active transposable elements that can efficiently be used for the generation of large numbers of insertion mutations, while some other species lack such options. Mutant plants produced by Agrobacterium or transposon mutagenesis and having altered expression of a polypeptide of interest can be identified using the polynucleotides of the present invention. For example, a large population of mutated plants may be screened with polynucleotides encoding the polypeptide of interest to detect mutated plants having an insertion in the gene encoding the polypeptide of interest.

Transgenic plants may comprise a stack of one or more polynucleotides disclosed herein resulting in the production or suppression of multiple polypeptide sequences. Transgenic plants comprising stacks of polynucleotide sequences can be obtained by either or both of traditional breeding methods or through genetic engineering methods. These methods include, but are not limited to, breeding individual lines each comprising a polynucleotide of interest, transforming a transgenic plant comprising a gene disclosed herein with a subsequent gene, and co-transformation of genes into a single plant cell. Co-transformation of genes can be carried out using single transformation vectors comprising multiple genes or genes carried separately on multiple vectors.

Transgenic plants comprising or derived from plant cells of this invention transformed with recombinant DNA can be further enhanced with stacked traits, e.g. a crop plant having an enhanced trait resulting from expression of DNA disclosed herein in combination with herbicide and/or pest resistance traits. For example, genes of the current invention can be stacked with other traits of agronomic interest, such as a trait providing herbicide resistance, or insect resistance, such as using a gene from Bacillus thuringensis to provide resistance against lepidopteran, coliopteran, homopteran, hemiopteran, and other insects. Herbicides for which transgenic plant tolerance has been demonstrated and the method of the present invention can be applied include, but are not limited to, glyphosate, dicamba, glufosinate, sulfonylurea, bromoxynil and norflurazon herbicides. Polynucleotide molecules encoding proteins involved in herbicide tolerance are well-known in the art and include, but are not limited to, a polynucleotide molecule encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) disclosed in U.S. Pat. Nos. 5,094,945; 5,627,061; 5,633,435 and 6,040,497 for imparting glyphosate tolerance; polynucleotide molecules encoding a glyphosate oxidoreductase (GOX) disclosed in U.S. Pat. No. 5,463,175 and a glyphosate-N-acetyl transferase (GAT) disclosed in US Patent Application Publication 2003/0083480 A1 also for imparting glyphosate tolerance; dicamba monooxygenase disclosed in US Patent Application Publication 2003/0135879 A1 for imparting dicamba tolerance; a polynucleotide molecule encoding bromoxynil nitrilase (Bxn) disclosed in U.S. Pat. No. 4,810,648 for imparting bromoxynil tolerance; a polynucleotide molecule encoding phytoene desaturase (crtI) described in Misawa et al, (1993) Plant J. 4:833-840 and in Misawa et al, (1994) Plant J. 6:481-489 for norflurazon tolerance; a polynucleotide molecule encoding acetohydroxyacid synthase (AHAS, aka ALS) described in Sathasiivan et al. (1990) Nucl. Acids Res. 18:2188-2193 for imparting tolerance to sulfonylurea herbicides; polynucleotide molecules known as bar genes disclosed in DeBlock, et al. (1987) EMBO J. 6:2513-2519 for imparting glufosinate and bialaphos tolerance; polynucleotide molecules disclosed in US Patent Application Publication 2003/010609 A1 for imparting N-amino methyl phosphonic acid tolerance; polynucleotide molecules disclosed in U.S. Pat. No. 6,107,549 for imparting pyridine herbicide resistance; molecules and methods for imparting tolerance to multiple herbicides such as glyphosate, atrazine, ALS inhibitors, isoxoflutole and glufosinate herbicides are disclosed in U.S. Pat. No. 6,376,754 and US Patent Application Publication 2002/0112260. Molecules and methods for imparting insect/nematode/virus resistance are disclosed in U.S. Pat. Nos. 5,250,515; 5,880,275; 6,506,599; 5,986,175 and US Patent Application Publication 2003/0150017 A1.

Plant Cell Transformation Methods

Numerous methods for transforming chromosomes in a plant cell nucleus with recombinant DNA are known in the art and are used in methods of preparing a transgenic plant cell nucleus cell, and plant. Two effective methods for such transformation are Agrobacterium-mediated transformation and microprojectile bombardment. Microprojectile bombardment methods are illustrated in U.S. Pat. Nos. 5,015,580 (soybean); 5,550,318 (corn); 5,538,880 (corn); 5,914,451 (soybean); 6,160,208 (corn); 6,399,861 (corn); 6,153,812 (wheat) and 6,365,807 (rice) and Agrobacterium-mediated transformation is described in U.S. Pat. Nos. 5,159,135 (cotton); 5,824,877 (soybean); 5,463,174 (canola); 5,591,616 (corn); 5,846,797 (cotton); 6,384,301 (soybean), 7,026,528 (wheat) and 6,329,571 (rice), US Patent Application Publication 2004/0087030 A1 (cotton), and US Patent Application Publication 2001/0042257 A1 (sugar beet), all of which are incorporated herein by reference for enabling the production of transgenic plants. Transformation of plant material is practiced in tissue culture on a nutrient media, i.e. a mixture of nutrients that will allow cells to grow in vitro. Recipient cell targets include, but are not limited to, meristem cells, hypocotyls, calli, immature embryos and gametic cells such as microspores, pollen, sperm and egg cells. Callus may be initiated from tissue sources including, but not limited to, immature embryos, hypocotyls, seedling apical meristems, microspores and the like. Cells containing a transgenic nucleus are grown into transgenic plants.

In addition to direct transformation of a plant material with a recombinant DNA, a transgenic plant cell nucleus can be prepared by crossing a first plant having cells with a transgenic nucleus with recombinant DNA with a second plant lacking the transgenic nucleus. For example, recombinant DNA can be introduced into a nucleus from a first plant line that is amenable to transformation to transgenic nucleus in cells that are grown into a transgenic plant which can be crossed with a second plant line to introgress the recombinant DNA into the second plant line. A transgenic plant with recombinant DNA providing an enhanced trait, e.g. enhanced yield, can be crossed with transgenic plant line having other recombinant DNA that confers another trait, for example herbicide resistance or pest resistance, to produce progeny plants having recombinant DNA that confers both traits. Typically, in such breeding for combining traits the transgenic plant donating the additional trait is a male line and the transgenic plant carrying the base traits is the female line. The progeny of this cross will segregate such that some of the plants will carry the DNA for both parental traits and some will carry DNA for one parental trait; such plants can be identified by markers associated with parental recombinant DNA, e.g. marker identification by analysis for recombinant DNA or, in the case where a selectable marker is linked to the recombinant, by application of the selecting agent such as a herbicide for use with a herbicide tolerance marker, or by selection for the enhanced trait. Progeny plants carrying DNA for both parental traits can be crossed back into the female parent line multiple times, for example usually 6 to 8 generations, to produce a progeny plant with substantially the same genotype as one original transgenic parental line but for the recombinant DNA of the other transgenic parental line

In the practice of transformation DNA is typically introduced into only a small percentage of target plant cells in any one transformation experiment. Marker genes are used to provide an efficient system for identification of those cells that are stably transformed by receiving and integrating a recombinant DNA molecule into their genomes. Preferred marker genes provide selective markers which confer resistance to a selective agent, such as an antibiotic or a herbicide. Any of the herbicides to which plants of this invention may be resistant are useful agents for selective markers. Potentially transformed cells are exposed to the selective agent. In the population of surviving cells will be those cells where, generally, the resistance-conferring gene is integrated and expressed at sufficient levels to permit cell survival. Cells may be tested further to confirm stable integration of the exogenous DNA. Commonly used selective marker genes include those conferring resistance to antibiotics such as kanamycin and paromomycin (nptII), hygromycin B (aph IV), spectinomycin (aadA) and gentamycin (aac3 and aacC4) or resistance to herbicides such as glufosinate (bar or pat), dicamba (DMO) and glyphosate (aroA or EPSPS). Examples of such selectable markers are illustrated in U.S. Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and 6,118,047. Markers which provide an ability to visually screen transformants can also be employed, for example, a gene expressing a colored or fluorescent protein such as a luciferase or green fluorescent protein (GFP) or a gene expressing a beta-glucuronidase or uidA gene (GUS) for which various chromogenic substrates are known.

Plant cells that survive exposure to the selective agent, or plant cells that have been scored positive in a screening assay, may be cultured in regeneration media and allowed to mature into plants. Developing plantlets regenerated from transformed plant cells can be transferred to plant growth mix, and hardened off, for example, in an environmentally controlled chamber at about 85% relative humidity, 600 ppm CO₂, and 25-250 microeinsteins m⁻² s⁻¹ of light, prior to transfer to a greenhouse or growth chamber for maturation. Plants are regenerated from about 6 weeks to 10 months after a transformant is identified, depending on the initial tissue, and plant species. Plants may be pollinated using conventional plant breeding methods known to those of skill in the art and seed produced, for example self-pollination is commonly used with transgenic corn. The regenerated transformed plant or its progeny seed or plants can be tested for expression of the recombinant DNA and selected for the presence of enhanced agronomic trait.

Transgenic Plants and Seeds

Transgenic plants derived from transgenic plant cells having a transgenic nucleus of this invention are grown to generate transgenic plants having an enhanced trait as compared to a control plant and produce transgenic seed and haploid pollen of this invention. Such plants with enhanced traits are identified by selection of transformed plants or progeny seed for the enhanced trait. For efficiency a selection method is designed to evaluate multiple transgenic plants (events) comprising the recombinant DNA, for example multiple plants from 2 to 20 or more transgenic events. Transgenic plants grown from transgenic seed provided herein demonstrate improved agronomic traits that contribute to increased yield or other trait that provides increased plant value, including, for example, improved seed quality. Of particular interest are plants having enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil. Table 1 provides a list of protein encoding DNA (“genes”) that are useful as recombinant DNA for production of transgenic plants with enhanced agronomic trait, the elements of Table 1 are described by reference to:

“PEP SEQ ID NO” identifies an amino acid sequence from SEQ ID NO: 11 to 20. “NUC SEQ ID NO” identifies a DNA sequence from SEQ ID NO:1 to 10. “Gene ID” refers to an arbitrary identifier. “Gene Name” denotes a common name for protein encoded by the recombinant DNA. “Annotation” refers to a description of the top hit protein obtained from an amino acid sequence query of each PEP SEQ ID NO to GENBANK database of the National Center for Biotechnology Information (ncbi).

TABLE 1 NUC PEP SEQ ID SEQ ID NO NO Gene ID Gene Name Annotation 1 11 Mnom000034 Corn ZmLac gb|AAX83113.1|laccase 1 [Zea mays] 2 12 Mnom000037 Anabaena FLD gb|AAB20462.1|flavodoxin [Anabaena] 3 13 Mnom000048 Corn TPS1 gb|EAY98715.1|hypothetical protein OsI_019948 [Oryza sativa (indica cultivar-group)] 4 14 Mnom000049 Corn TPP1 gb|EAY75823.1|hypothetical protein OsI_003670 [Oryza sativa (indica cultivar-group)] 5 15 Mnom000067 Lycopersicon AlaT gb|AAZ43369.1|AlaT1 [Vitis vinifera] 6 16 Mnom000068 Lycopersicon AlaT gb|AAZ43369.1|AlaT1 [Vitis vinifera] 7 17 Mnom000090 Corn ZmGS3 gb|ABC84855.1|grain length and weight protein [Oryza sativa (indica cultivar-group)] 8 18 Mnom000091 Corn ZmGS3 gb|ABC84855.1|grain length and weight protein [Oryza sativa (indica cultivar-group)] 9 19 Mnom000092 Corn ZmBB gb|EAZ25768.1|hypothetical protein OsJ_009251 [Oryza sativa (japonica cultivar-group)] 10 20 Mnom000095 Corn ref|NP_001052879.1|Os04g0442000 [Oryza sativa OSJNBa0064D (japonica cultivar-group)] [Mendel G471-like ner] 20.11 Protein Selection Methods for Transgenic Plants with Enhanced Agronomic Trait

Within a population of transgenic plants each regenerated from a plant cell having a nucleus with recombinant DNA many plants that survive to fertile transgenic plants that produce seeds and progeny plants will not exhibit an enhanced agronomic trait. Selection from the population is necessary to identify one or more transgenic plant cells having a transgenic nucleus that can provide plants with the enhanced trait. Transgenic plants having enhanced traits are selected from populations of plants regenerated or derived from plant cells transformed as described herein by evaluating the plants in a variety of assays to detect an enhanced trait, e.g. enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil. These assays also may take many forms including, but not limited to, direct screening for the trait in a greenhouse or field trial or by screening for a surrogate trait. Such analyses can be directed to detecting changes in the chemical composition, biomass, physiological properties, morphology of the plant. Changes in chemical compositions such as nutritional composition of grain can be detected by analysis of the seed composition and content of protein, free amino acids, oil, free fatty acids, starch or tocopherols. Changes in biomass characteristics can be made on greenhouse or field grown plants and can include plant height, stem diameter, root and shoot dry weights; and, for corn plants, ear length and diameter. Changes in physiological properties can be identified by evaluating responses to stress conditions, for example assays using imposed stress conditions such as water deficit, nitrogen deficiency, cold growing conditions, pathogen or insect attack or light deficiency, or increased plant density. Changes in morphology can be measured by visual observation of tendency of a transformed plant with an enhanced agronomic trait to also appear to be a normal plant as compared to changes toward bushy, taller, thicker, narrower leaves, striped leaves, knotted trait, chlorosis, albino, anthocyanin production, or altered tassels, ears or roots. Other selection properties include days to pollen shed, days to silking, leaf extension rate, chlorophyll content, leaf temperature, stand, seedling vigor, internode length, plant height, leaf number, leaf area, tillering, brace roots, stay green, stalk lodging, root lodging, plant health, barreness/prolificacy, green snap, and pest resistance. In addition, phenotypic characteristics of harvested grain may be evaluated, including number of kernels per row on the ear, number of rows of kernels on the ear, kernel abortion, kernel weight, kernel size, kernel density and physical grain quality.

Assays for screening for a desired trait are readily designed by those practicing in the art. The following illustrates useful screening assays for corn traits using hybrid corn plants. The assays can be readily adapted for screening other plants such as canola, cotton and soybean either as hybrids or inbreds.

Transgenic corn plants having nitrogen use efficiency are identified by screening in fields with three levels of nitrogen (N) fertilizer being applied, e.g. low level (0 N), medium level (80 lb/ac) and high level (180 lb/ac). Plants with enhanced nitrogen use efficiency provide higher yield as compared to control plants.

Transgenic corn plants having enhanced yield are identified by screening using progeny of the transgenic plants over multiple locations with plants grown under optimal production management practices and maximum weed and pest control. A useful target for improved yield is a 5% to 10% increase in yield as compared to yield produced by plants grown from seed for a control plant. Selection methods may be applied in multiple and diverse geographic locations, for example up to 16 or more locations, over one or more planting seasons, for example at least two planting seasons, to statistically distinguish yield improvement from natural environmental effects.

Transgenic corn plants having enhanced water use efficiency are identified by screening plants in an assay where water is withheld for a period to induce stress followed by watering to revive the plants. For example, a useful selection process imposes 3 drought/re-water cycles on plants over a total period of 15 days after an initial stress free growth period of 11 days. Each cycle consists of 5 days, with no water being applied for the first four days and a water quenching on the 5th day of the cycle. The primary phenotypes analyzed by the selection method are the changes in plant growth rate as determined by height and biomass during a vegetative drought treatment.

Transgenic corn plants having enhanced cold tolerance are identified by screening plants in a cold germination assay and/or a cold tolerance field trial. In a cold germination assay trays of transgenic and control seeds are placed in a growth chamber at 9.7° C. for 24 days (no light). Seeds having higher germination rates as compared to the control are identified as having enhanced cold tolerance. In a cold tolerance field trial plants with enhanced cold tolerance are identified from field planting at an earlier date than conventional Spring planting for the field location. For example, seeds are planted into the ground around two weeks before local farmers begin to plant corn so that a significant cold stress is exerted onto the crop, named as cold treatment. Seeds also are planted under local optimal planting conditions such that the crop has little or no exposure to cold condition, named as normal treatment. At each location, seeds are planted under both cold and normal conditions preferably with multiple repetitions per treatment.

Transgenic corn plants having seeds with increased protein and/or oil levels are identified by analyzing progeny seed for protein and/or oil. Near-infrared transmittance spectrometry is a non-destructive, high-throughput method that is useful to determine the composition of a bulk seed sample for properties listed in table 2.

TABLE 2 Typical sample(s): Whole grain corn and soybean seeds Typical analytical range: Corn - moisture 5-15%, oil 5-20%, protein 5-30%, starch 50-75%, and density 1.0-1.3%. Soybean - moisture 5-15%, oil 15-25%, and protein 35-50%.

Although the plant cells and methods of this invention can be applied to any plant cell, plant, seed or pollen, e.g. any fruit, vegetable, grass, tree or ornamental plant, the various aspects of the invention are preferably applied to corn, soybean, cotton, canola, alfalfa, wheat, rice, sugarcane, and sugar beet plants. In many cases the invention is applied to corn plants that are inherently resistant to disease from the Mal de Rio Cuarto virus or the Puccina sorghi fungus or both.

The following examples are included to demonstrate aspects of the invention, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar results without departing from the spirit and scope of the invention.

Example 1 Plant Expression Constructs

This example illustrates the construction of plasmids for transferring recombinant DNA into a plant cell nucleus that can be regenerated into transgenic plants.

A. Plant Expression Constructs for Corn Transformation

A base corn transformation vector pMON93039, as set forth in SEQ ID NO:1352, illustrated in Table 3 and FIG. 1, is fabricated for use in preparing recombinant DNA for Agrobacterium-mediated transformation into corn tissue.

TABLE 3 Coordinates of Function Name Annotation SEQ ID NO: 1352 Agrobacterium B-AGRtu.right border Agro right border sequence, 11364-11720 T-DNA transfer essential for transfer of T-DNA. Gene of interest E-Os.Act1 Upstream promoter region of the  19-775 expression rice actin 1 gene cassette E-CaMV.35S.2xA1- Duplicated35S A1-B3 domain  788-1120 B3 without TATA box P-Os.Act1 Promoter region of the rice actin 1 1125-1204 gene L-Ta.Lhcb1 5′ untranslated leader of wheat 1210-1270 major chlorophyll a/b binding protein I-Os.Act1 First intron and flanking UTR exon 1287-1766 sequences from the rice actin 1 gene T-St.Pis4 3′ non-translated region of the 1838-2780 potato proteinase inhibitor II gene which functions to direct polyadenylation of the mRNA Plant selectable P-Os.Act1 Promoter from the rice actin 1 gene 2830-3670 marker L-Os.Act1 First exon of the rice actin 1 gene 3671-3750 expression I-Os.Act1 First intron and flanking UTR exon 3751-4228 cassette sequences from the rice actin 1 gene TS-At.ShkG-CTP2 Transit peptide region of 4238-4465 Arabidopsis EPSPS CR-AGRtu.aroA- Coding region for bacterial strain 4466-5833 CP4.nat CP4 native aroA gene. T-AGRtu.nos A 3′ non-translated region of the 5849-6101 nopaline synthase gene of Agrobacterium tumefaciens Ti plasmid which functions to direct polyadenylation of the mRNA. Agrobacterium B-AGRtu.left border Agro left border sequence, essential 6168-6609 T-DNA transfer for transfer of T-DNA. Maintenance in OR-Ec.oriV-RK2 The vegetative origin of replication 6696-7092 E. coli from plasmid RK2. CR-Ec.rop Coding region for repressor of 8601-8792 primer from the ColE1 plasmid. Expression of this gene product interferes with primer binding at the origin of replication, keeping plasmid copy number low. OR-Ec.ori-ColE1 The minimal origin of replication 9220-9808 from the E. coli plasmid ColE1. P-Ec.aadA-SPC/STR Promoter for Tn7 10339-10380 adenylyltransferase (AAD(3″)) CR-Ec.aadA- Coding region for Tn7 10381-11169 SPC/STR adenylyltransferase (AAD(3″)) conferring spectinomycin and streptomycin resistance. T-Ec.aadA-SPC/STR 3′ UTR from the Tn7 11170-11227 adenylyltransferase (AAD(3″)) gene of E. coli.

To construct transformation vectors for expressing a protein identified in Table 1, primers for PCR amplification of the protein coding nucleotides are designed at or near the start and stop codons of the coding sequence, in order to eliminate most of the 5′ and 3′ untranslated regions. The protein coding nucleotides are inserted into the base vector in the gene of interest expression cassette at an insertion site, i.e. between the intron element (coordinates 1287-1766) and the polyadenylation element (coordinates 1838-2780).

To construct transformation vectors for suppressing a protein identified in Table 1, the amplified protein coding nucleotides are assembled in a sense and antisense arrangement and inserted into the base vector at the insertion site in the gene of interest expression cassette to provide transcribed RNA that will form a double-stranded RNA for RNA interference suppression of the protein. In the embodiments of this invention the proteins that are suppressed are ZmLac, ZmGS3, and ZmBB.

B. Plant Expression Constructs for Soy and Canola Transformation

Vectors for use in transformation of soybean and canola tissue are prepared having the elements of expression vector pMON82053 (SEQ ID NO: 1353) as shown in Table 4 below and FIG. 2.

TABLE 4 Coordinates of Function Name Annotation SEQ ID NO: 1353 Agrobacterium T- B-AGRtu.left border Agro left border sequence, essential for 6144-6585 DNA transfer transfer of T-DNA. Plant selectable P-At.Act7 Promoter from the Arabidopsis actin 7 gene 6624-7861 marker expression L-At.Act7 5′UTR of Arabidopsis Act7 gene cassette I-At.Act7 Intron from the Arabidopsis actin7 gene TS-At.ShkG-CTP2 Transit peptide region of Arabidopsis 7864-8091 EPSPS CR-AGRtu.aroA- Synthetic CP4 coding region with dicot 8092-9459 CP4.nno_At preferred codon usage. T-AGRtu.nos A 3′ non-translated region of the nopaline 9466-9718 synthase gene of Agrobacterium tumefaciens Ti plasmid which functions to direct polyadenylation of the mRNA. Gene of interest P-CaMV.35S-enh Promoter for 35S RNA from CaMV  1-613 expression cassette containing a duplication of the −90 to −350 region. T-Gb.E6-3b 3′ untranslated region from the fiber protein  688-1002 E6 gene of sea-island cotton. Agrobacterium T- B-AGRtu.right Agro right border sequence, essential for 1033-1389 DNA transfer border transfer of T-DNA. Maintenance in E. coli OR-Ec.oriV-RK2 The vegetative origin of replication from 5661-6057 plasmid RK2. CR-Ec.rop Coding region for repressor of primer from 3961-4152 the ColE1 plasmid. Expression of this gene product interferes with primer binding at the origin of replication, keeping plasmid copy number low. OR-Ec.ori-ColE1 The minimal origin of replication from the 2945-3533 E. coli plasmid ColE1. P-Ec.aadA-SPC/STR Promoter for Tn7 adenylyltransferase 2373-2414 (AAD(3″)) CR-Ec.aadA- Coding region for Tn7 adenylyltransferase 1584-2372 SPC/STR (AAD(3″)) conferring spectinomycin and streptomycin resistance. T-Ec.aadA-SPC/STR 3′ UTR from the Tn7 adenylyltransferase 1526-1583 (AAD(3″)) gene of E. coli.

To construct transformation vectors for expressing a protein identified in Table 1, primers for PCR amplification of the protein coding nucleotides are designed at or near the start and stop codons of the coding sequence, in order to eliminate most of the 5′ and 3′ untranslated regions. The protein coding nucleotides are inserted into the base vector in the gene of interest expression cassette at an insertion site, i.e. between the promoter element (coordinates 1-613) and the polyadenylation element (coordinates 688-1002).

To construct transformation vectors for suppressing a protein identified in Table 1, the amplified protein coding nucleotides are assembled in a sense and antisense arrangement and inserted into the base vector at the insertion site in the gene of interest expression cassette to provide transcribed RNA that will form a double-stranded RNA for RNA interference suppression of the protein. In the embodiments of this invention the proteins that are suppressed are ZmLac, ZmGS3, and ZmBB.

C. Cotton Transformation Vector

Plasmids for use in transformation of cotton tissue are prepared with elements of expression vector pMON99053 (SEQ ID NO: 1354) as shown in Table 5 below and FIG. 3.

TABLE 5 Coordinates of SEQ ID NO: Function Name Annotation 1354 Agrobacterium T- B-AGRtu.right border Agro right border sequence, essential for  1-357 DNA transfer transfer of T-DNA. Gene of interest Exp-CaMV.35S- Enhanced version of the 35S RNA promoter  388-1091 expression cassette enh+Ph.DnaK from CaMV plus the petunia hsp70 5′ untranslated region T-Ps.RbcS2-E9 The 3′ non-translated region of the pea RbcS2 1165-1797 gene which functions to direct polyadenylation of the mRNA. Plant selectable Exp-CaMV.35S Promoter and 5′ untranslated region from the 1828-2151 marker expression 35S RNA of CaMV cassette CR-Ec.nptII-Tn5 Coding region for neomycin 2185-2979 phosphotransferase gene from transposon Tn5 which confers resistance to neomycin and kanamycin. T-AGRtu.nos A 3′ non-translated region of the nopaline 3011-3263 synthase gene of Agrobacterium tumefaciens Ti plasmid which functions to direct polyadenylation of the mRNA. Agrobacterium T- B-AGRtu.left border Agro left border sequence, essential for 3309-3750 DNA transfer transfer of T-DNA. Maintenance in E. coli OR-Ec.oriV-RK2 The vegetative origin of replication from 3837-4233 plasmid RK2. CR-Ec.rop Coding region for repressor of primer from 5742-5933 the ColE1 plasmid. Expression of this gene product interferes with primer binding at the origin of replication, keeping plasmid copy number low. OR-Ec.ori-ColE1 The minimal origin of replication from the E. coli 6361-6949 plasmid ColE1. P-Ec.aadA-SPC/STR Promoter for Tn7 adenylyltransferase 7480-7521 (AAD(3″)) CR-Ec.aadA-SPC/STR Coding region for Tn7 adenylyltransferase 7522-8310 (AAD(3″)) conferring spectinomycin and streptomycin resistance. T-Ec.aadA-SPC/STR 3′ UTR from the Tn7 adenylyltransferase 8311-8368 (AAD(3″)) gene of E. coli.

To construct transformation vectors for expressing a protein identified in Table 1, primers for PCR amplification of the protein coding nucleotides are designed at or near the start and stop codons of the coding sequence, in order to eliminate most of the 5′ and 3′ untranslated regions. The protein coding nucleotides are inserted into the base vector in the gene of interest expression cassette at an insertion site, i.e. between the promoter element (coordinates 388-1091) and the polyadenylation element (coordinates 1165-1797).

To construct transformation vectors for suppressing a protein identified in Table 1, the amplified protein coding nucleotides are assembled in a sense and antisense arrangement and inserted into the base vector at the insertion site in the gene of interest expression cassette to provide transcribed RNA that will form a double-stranded RNA for RNA interference suppression of the protein. In the embodiments of this invention the proteins that are suppressed are ZmLac, ZmGS3, and ZmBB.

D. Plant Expression Constructs for Gene Stacking in Corn.

A base corn transformation vector pMON96782, as set forth in SEQ ID NO: 1355, illustrated in Table 6 and FIG. 4, is fabricated for use in preparing recombinant DNA for Agrobacterium-mediated transformation into corn tissue.

TABLE 6 Coordinates of Function Name Annotation SEQ ID NO: 1355 Agrobacterium B-AGRtu.right border Agro right border sequence,  1-357 T-DNA transfer essential for transfer of T-DNA. Gene of interest P-Os.Act1 Promoter region of the rice actin 1  403-1243 expression gene cassette 1 L-Os.Act1 5′ untranslated leader of rice actin 1 1244-1323 gene I-Os.Act1 First intron and flanking UTR exon 1324-1801 sequences from the rice actin 1 gene T-Ta.Hsp17 3′ un-translated region of wheat low 1834-2043 molecular weight heat shock protein gene Gene of interest E-Os.Act1 Upstream Promoter region of rice 2136-2892 expression actin 1 cassette 2 E-CaMV.35S.2xA1- 35S A1-B3 Domain 2905-2937 B3 P-Os.Act1 Promoter from rice actin gene 3242-3321 L-Ta.Lhcb1 5′ untranslated leader from wheat 3327-3387 chlorophyll protein I-Os.Act1 Intron and 5′ untranslated region 3404-3883 from rice actin 1 gene T-AGRtu.tr7 3′ untranslated region from 3918-4425 “transcript 7” of Agrobacterium Plant selectable P.Os.TubA Promoter of alpha-tubulin gene of 4452-5650 marker rice expression L.Os.TubA 5′ untranslated region of an alpha 5651-5736 cassette tubulin from rice I.Os.TubA Intron 1 of an alpha tubulin from 5737-6632 rice. Ts.Ta.waxy.nno_Zm Chloroplast transit peptide from 6637-6846 wheat starch synthase Cr.AGRtu.aroA- CP4 EPSPS gene 6847-8214 CP4.nno_Zm T.Os.TubA 3′ untranslated region of alpha 8219-8800 tubulin from rice Agrobacterium B-AGRtu.left border Left border sequence for T-DNA 8828-9269 T-DNA transfer transfer Maintenance in OR-Ec.oriV-RK2 Origin of replication from the E. coli 9356-9752 E. coli plasmid RK2. Cr-Ec.rop Coding region for repressor of 11261-11452 primer from ColE1 plasmid OR-Ec.ori-ColE1 Minimum origin of replication from 11880-12468 E. coli colE1 plasmid. P-Ec.aadA-SPC/STR Promoter for Tn7 12999-13040 adenylyltransferase gene CR-Ec.aadA- Coding region for Tn7 13041-13829 SPC/STR adenylyltransferase gene T-Ec.aadA-SPC/STR 3′ untranslated region from Tn7 13830-13887 adenylyltransferase gene

Primers for PCR amplification of protein coding nucleotides of the genes of interest are designed at or near the start and stop codons of the coding sequence, in order to eliminate most of the 5′ and 3′ untranslated regions. Protein coding regions of genes encoding a first and second protein of interest are amplified. The amplified region from the first gene of interest is cloned between nucleotides 1801 and 1834 of the base vector and the amplified region from the second gene of interest is cloned between nucleotides 3883 and 3918 of the base vector.

Example 2 Corn Transformation

This example illustrates transformation methods useful in producing a transgenic nucleus in a corn plant cell, and the plants, seeds and pollen produced from a transgenic cell with such a nucleus having an enhanced trait, i.e. enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil. A plasmid vector is prepared by cloning the DNA of SEQ ID NO:1 into the gene of interest expression cassette in the base vector for use in corn transformation of corn tissue provided in Example 1, Table 3.

For Agrobacterium-mediated transformation of corn embryo cells corn plants of a readily transformable line are grown in the greenhouse and ears are harvested when the embryos are 1.5 to 2.0 mm in length. Ears are surface sterilized by spraying or soaking the ears in 80% ethanol, followed by air drying. Immature embryos are isolated from individual kernels on surface sterilized ears. Prior to inoculation of maize cells, Agrobacterium cells are grown overnight at room temperature. Immature maize embryo cells are inoculated with Agrobacterium shortly after excision, and incubated at room temperature with Agrobacterium for 5-20 minutes. Immature embryo plant cells are then co-cultured with Agrobacterium for 1 to 3 days at 23° C. in the dark. Co-cultured embryos are transferred to selection media and cultured for approximately two weeks to allow embryogenic callus to develop. Embryogenic callus is transferred to culture medium containing 100 mg/L paromomycin and subcultured at about two week intervals. Transformed plant cells are recovered 6 to 8 weeks after initiation of selection.

For Agrobacterium-mediated transformation of maize callus immature embryos are cultured for approximately 8-21 days after excision to allow callus to develop. Callus is then incubated for about 30 minutes at room temperature with the Agrobacterium suspension, followed by removal of the liquid by aspiration. The callus and Agrobacterium are co-cultured without selection for 3-6 days followed by selection on paromomycin for approximately 6 weeks, with biweekly transfers to fresh media. Paromomycin resistant calli are identified about 6-8 weeks after initiation of selection.

To regenerate transgenic corn plants a callus of transgenic plant cells resulting from transformation and selection is placed on media to initiate shoot development into plantlets which are transferred to potting soil for initial growth in a growth chamber at 26° C. followed by a mist bench before transplanting to 5 inch pots where plants are grown to maturity. The regenerated plants are self-fertilized and seed is harvested for use in one or more methods to select seeds, seedlings or progeny second generation transgenic plants (R2 plants) or hybrids, e.g. by selecting transgenic plants exhibiting an enhanced trait as compared to a control plant.

The above process is repeated to produce multiple events of transgenic corn plant cells that are transformed with recombinant DNA from each of the genes identified in Table 1. Events are designed to produce in the transgenic cells one of the proteins identified in Table 1, except the proteins of SEQ ID NOs: 11 and 17-19 which are suppressed. Progeny transgenic plants and seed of the transformed plant cells are screened for enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil. From each group of multiple events of transgenic plants with a specific recombinant DNA from Table 1 the event that produces the greatest enhancement in yield, water use efficiency, nitrogen use efficiency, enhanced cold tolerance, enhanced seed protein and enhanced seed oil is identified and progeny seed is selected for commercial development.

Example 3 Soybean Transformation

This example illustrates plant transformation useful in producing a transgenic nucleus in a soybean plant cell, and the plants, seeds and pollen produced from a transgenic cell with such a nucleus having an enhanced trait, i.e. enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil.

For Agrobacterium mediated transformation, soybean seeds are imbided overnight and the meristem explants excised. The explants are placed in a wounding vessel. Soybean explants and induced Agrobacterium cells from a strain containing plasmid DNA with the gene of interest cassette and a plant selectable marker cassette are mixed no later than 14 hours from the time of initiation of seed imbibition, and wounded using sonication. Following wounding, explants are placed in co-culture for 2-5 days at which point they are transferred to selection media for 6-8 weeks to allow selection and growth of transgenic shoots. Resistant shoots are harvested approximately 6-8 weeks and placed into selective rooting media for 2-3 weeks. Shoots producing roots are transferred to the greenhouse and potted in soil. Shoots that remain healthy on selection, but do not produce roots are transferred to non-selective rooting media for an additional two weeks. Roots from any shoots that produce roots off selection are tested for expression of the plant selectable marker before they are transferred to the greenhouse and potted in soil.

The above process is repeated to produce multiple events of transgenic soybean plant cells that are transformed with recombinant DNA from each of the genes identified in Table 1. Events are designed to produce in the transgenic cells one of the proteins identified in Table 1, except the proteins of SEQ ID NOs: 11, and 17-19 which are suppressed. Progeny transgenic plants and seed of the transformed plant cells are screened for enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced seed protein and enhanced seed oil. From each group of multiple events of transgenic plants with a specific recombinant DNA from Table 1 the event that produces the greatest enhancement in yield, water use efficiency, nitrogen use efficiency, enhanced cold tolerance, enhanced seed protein and enhanced seed oil is identified and progeny seed is selected for commercial development.

Example 4 Cotton Transgenic Plants with Enhanced Agronomic Traits

This example illustrates plant transformation useful in producing a transgenic nucleus in a cotton plant cell, and the plants, seeds and pollen produced from a transgenic cell with such a nucleus having an enhanced trait, i.e. enhanced water use efficiency, increased yield, enhanced nitrogen use efficiency and enhanced seed oil.

Transgenic cotton plants containing each recombinant DNA having a sequence of SEQ ID NO: 1 through SEQ ID NO: 10 are obtained by transforming with recombinant DNA from each of the genes identified in Table 1 using Agrobacterium-mediated transformation. The above process is repeated to produce multiple events of transgenic cotton plant cells that are transformed with recombinant DNA from each of the genes identified in Table 1. Events are designed to produce in the transgenic cells one of the proteins identified in Table 1, except the proteins of SEQ ID NOs: 11, and 17-19 which are suppressed.

From each group of multiple events of transgenic plants with a specific recombinant DNA from Table 1 the event that produces the greatest enhancement in yield, water use efficiency, nitrogen use efficiency, enhanced cold tolerance, enhanced seed protein and enhanced seed oil is identified and progeny seed is selected for commercial development.

Progeny transgenic plants are selected from a population of transgenic cotton events under specified growing conditions and are compared with control cotton plants. Control cotton plants are substantially the same cotton genotype but without the recombinant DNA, for example, either a parental cotton plant of the same genotype that was not transformed with the identical recombinant DNA or a negative isoline of the transformed plant. Additionally, a commercial cotton cultivar adapted to the geographical region and cultivation conditions, i.e. cotton variety ST474, cotton variety FM 958, and cotton variety Siokra L-23, are used to compare the relative performance of the transgenic cotton plants containing the recombinant DNA.

Transgenic cotton plants with enhanced yield and water use efficiency are identified by growing under variable water conditions. Specific conditions for cotton include growing a first set of transgenic and control plants under “wet” conditions, i.e. irrigated in the range of 85 to 100 percent of evapotranspiration to provide leaf water potential of −14 to −18 bars, and growing a second set of transgenic and control plants under “dry” conditions, i.e. irrigated in the range of 40 to 60 percent of evapotranspiration to provide a leaf water potential of −21 to −25 bars. Pest control, such as weed and insect control is applied equally to both wet and dry treatments as needed. Data gathered during the trial includes weather records throughout the growing season including detailed records of rainfall; soil characterization information; any herbicide or insecticide applications; any gross agronomic differences observed such as leaf morphology, branching habit, leaf color, time to flowering, and fruiting pattern; plant height at various points during the trial; stand density; node and fruit number including node above white flower and node above crack boll measurements; and visual wilt scoring. Cotton boll samples are taken and analyzed for lint fraction and fiber quality. The cotton is harvested at the normal harvest timeframe for the trial area. Enhanced water use efficiency is indicated by increased yield, improved relative water content, enhanced leaf water potential, increased biomass, enhanced leaf extension rates, and improved fiber parameters.

Example 5 Canola Transformation

This example illustrates plant transformation useful in producing the transgenic canola plants of this invention and the production and identification of transgenic seed for transgenic canola having enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil.

Tissues from in vitro grown canola seedlings are prepared and inoculated with overnight-grown Agrobacterium cells containing plasmid DNA with the gene of interest cassette and a plant selectable marker cassette. Following co-cultivation with Agrobacterium, the infected tissues are allowed to grow on selection to promote growth of transgenic shoots, followed by growth of roots from the transgenic shoots. The selected plantlets are then transferred to the greenhouse and potted in soil. Molecular characterizations are performed to confirm the presence of the gene of interest, and its expression in transgenic plants and progenies. Progeny transgenic plants are selected from a population of transgenic canola events under specified growing conditions and are compared with control canola plants. Control canola plants are substantially the same canola genotype but without the recombinant DNA, for example, either a parental canola plant of the same genotype that is not transformed with the identical recombinant DNA or a negative isoline of the transformed plant.

Transgenic canola plant cells are transformed with each of the recombinant DNA identified in Table 1. The above process is repeated to produce multiple events of transgenic canola plant cells that are transformed with recombinant DNA from each of the genes identified in Table 1. Events are designed to produce in the transgenic cells one of the proteins identified in Table 1, except the proteins of SEQ ID NOs: 11, and 17-19 which are suppressed. Progeny transgenic plants and seed of the transformed plant cells are screened for enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced seed protein and enhanced seed oil. From each group of multiple events of transgenic plants with a specific recombinant DNA from Table 1 the event that produces the greatest enhancement in yield, water use efficiency, nitrogen use efficiency, enhanced cold tolerance, enhanced seed protein and enhanced seed oil is identified and progeny seed is selected for commercial development.

Example 6 Homolog Identification

This example illustrates the identification of homologs of proteins encoded by the DNA identified in Table 1 which is used to provide transgenic seed and plants having enhanced agronomic traits. From the sequence of the homologs, homologous DNA sequence can be identified for preparing additional transgenic seeds and plants of this invention with enhanced agronomic traits.

An “All Protein Database” was constructed of known protein sequences using a proprietary sequence database and the National Center for Biotechnology Information (NCBI) non-redundant amino acid database (nr.aa). For each organism from which a polynucleotide sequence provided herein was obtained, an “Organism Protein Database” was constructed of known protein sequences of the organism; it is a subset of the All Protein Database based on the NCBI taxonomy ID for the organism.

The All Protein Database was queried using amino acid sequences provided herein as SEQ ID NO: 11 through SEQ ID NO: 20 using NCBI “blastp” program with E-value cutoff of 1e-8. Up to 1000 top hits were kept, and separated by organism names. For each organism other than that of the query sequence, a list was kept for hits from the query organism itself with a more significant E-value than the best hit of the organism. The list contains likely duplicated genes of the polynucleotides provided herein, and is referred to as the Core List. Another list was kept for all the hits from each organism, sorted by E-value, and referred to as the Hit List.

The Organism Protein Database was queried using polypeptide sequences provided herein as SEQ ID NO: 11 through SEQ ID NO: 20 using NCBI “blastp” program with E-value cutoff of 1e-4. Up to 1000 top hits were kept. A BLAST searchable database was constructed based on these hits, and is referred to as “SubDB”. SubDB is queried with each sequence in the Hit List using NCBI “blastp” program with E-value cutoff of 1e-8. The hit with the best E-value was compared with the Core List from the corresponding organism. The hit is deemed a likely ortholog if it belongs to the Core List, otherwise it is deemed not a likely ortholog and there is no further search of sequences in the Hit List for the same organism. Homologs from a large number of distinct organisms were identified and are reported below in table 7 with the SEQ ID NO of the original query sequence and the identified homologs as [SEQ ID NO]: [Homolog SEQ ID NOs].

TABLE 7 Protein Sequences and their Homologs 11:    24   28   32   34   40   44   46   47   49   50   51   52   53   60   68   71   72   74   75   80   81   85   87   90  118  120  121  129  135  143  153  154  157  159  164  168  173  176  182  184  194  197  200  202  203  213  214  228  237  246  248  249  251  253  255  258  261  268  274  277  292  294  299  303  306  313  314  320  325  326  328  333  347  348  349  354  355  357  360  361  362  363  364  365  366  370  374  383  389  390  391  395  397  425  426  431  434  436  456  457  459  463  467  469  470  471  472  473  474  477  480  488  490  492  499  508  515  522  529  530  532  540  541  555  568  574  577  578  579  581  585  587  588  589  595  596  598  600  610  616  618  621  623  626  633  638  639  640  643  644  645  651  652  658  661  662  663  666  674  677  691  692  693  694  695  709  717  718  720  728  729  745  746  753  756  757  774  775  784  787  791  793  799  801  810  825  831  837  850  851  856  857  865  866  868  871  873  875  876  879  881  886  888  891  892  896  899  904  905  906  908  916  917  919  929  935  940  943  946  948  950  962  963  964  969  974  982  993  997 1005 1009 1011 1013 1015 1027 1028 1031 1032 1035 1049 1050 1059 1066 1068 1069 1076 1083 1085 1088 1090 1093 1095 1097 1098 1100 1107 1108 1110 1111 1117 1118 1120 1131 1134 1136 1137 1145 1146 1147 1149 1150 1153 1155 1156 1158 1168 1169 1170 1176 1184 1188 1193 1196 1205 1207 1212 1216 1226 1229 1230 1232 1233 1236 1241 1242 1243 1244 1248 1249 1252 1254 1256 1257 1260 1262 1265 1267 1269 1280 1283 1287 1292 1306 1307 1334 1335 1345 1350 12:    36   38   43   45   48   56   61   62   65   73   78   79   82   83   84   89  100  107  109  124  126  131  132  142  144  146  148  152  167  171  196  204  206  212  215  218  219  224  233  235  242  243  254  260  264  265  267  279  290  291  295  297  305  311  316  321  322  324  331  332  340  342  358  369  375  398  410  413  414  417  435  437  438  441  462  468  475  485  493  496  498  500  502  504  509  511  516  518  521  525  531  534  538  539  542  546  556  563  580  606  607  611  620  629  632  636  656  664  665  678  683  705  706  710  711  725  738  742  743  762  788  795  798  802  803  812  822  835  838  839  845  846  852  860  863  869  872  877  883  887  894  930  931  936  939  942  944  971  977  979  983  992  999 1007 1010 1030 1046 1048 1065 1075 1087 1091 1112 1113 1119 1124 1133 1135 1181 1182 1192 1195 1197 1206 1211 1213 1217 1219 1220 1223 1234 1235 1237 1239 1246 1247 1251 1258 1259 1261 1266 1300 1310 1317 1321 1328 1331 13:    23   30   33   42   54   55   63   64   66   88   92   93   94   95   96   97   99  103  104  106  114  115  117  119  127  128  130  138  147  150  151  155  158  160  165  166  175  178  190  191  195  198  208  210  211  221  229  230  238  247  252  256  257  259  262  263  266  272  283  287  317  327  329  330  353  356  368  378  379  385  387  394  399  404  409  411  419  427  428  433  439  440  444  446  449  458  460  461  481  487  503  505  507  510  513  527  537  545  548  550  552  553  557  558  559  564  565  569  572  584  593  602  605  608  609  612  617  619  625  637  647  650  660  672  690  696  703  716  722  726  730  731  735  747  749  750  755  761  766  771  776  779  786  790  794  804  806  811  817  821  824  826  827  828  829  836  841  847  848  854  867  870  878  884  893  895  897  907  909  910  914  918  923  925  928  933  934  951  952  954  955  957  958  959  965  975  985  987  988  991 1012 1019 1020 1022 1029 1033 1034 1036 1039 1041 1043 1044 1052 1056 1057 1060 1061 1063 1071 1072 1073 1084 1086 1094 1102 1115 1122 1126 1128 1144 1148 1151 1159 1161 1162 1173 1174 1177 1179 1189 1190 1201 1202 1203 1208 1221 1225 1227 1228 1231 1255 1270 1273 1278 1279 1291 1296 1301 1302 1318 1322 1324 1330 1338 1346 1348 14:    23   30   33   54   55   63   64   66   88   91   92   93   94   95   96   97   99  103  104  106  112  115  117  119  127  128  130  137  138  147  150  151  158  160  162  165  174  175  178  181  186  189  190  191  198  201  217  221  229  230  238  244  247  252  256  257  259  262  263  266  269  272  276  283  286  317  318  319  327  329  330  344  353  356  368  379  385  387  393  394  399  404  408  409  411  419  423  427  440  445  446  449  450  458  460  461  482  487  489  503  505  507  510  513  526  527  535  537  545  548  550  551  552  553  557  558  559  569  572  575  584  590  593  603  605  608  609  612  619  625  628  631  637  642  647  650  653  660  668  672  675  688  690  696  700  703  707  716  722  726  730  731  732  735  747  749  750  754  755  761  766  771  776  778  779  780  781  782  786  790  794  804  806  811  817  820  821  824  826  828  836  841  847  848  854  867  870  878  882  884  893  895  897  907  909  910  913  914  918  920  921  923  924  932  933  934  949  951  952  954  955  957  959  960  965  975  985  987  988  991 1012 1019 1020 1022 1026 1029 1033 1034 1036 1039 1041 1043 1044 1052 1057 1060 1061 1063 1071 1072 1073 1077 1084 1086 1094 1102 1109 1126 1128 1132 1138 1144 1148 1157 1159 1161 1162 1173 1174 1177 1179 1189 1190 1202 1203 1208 1221 1222 1225 1228 1231 1255 1270 1271 1273 1276 1284 1289 1291 1293 1301 1302 1318 1322 1324 1346 1348 15:    21   26   27   31   35   37   41   57   58   59   69   70   76   77   86   98  101  102  108  110  111  113  116  122  123  125  133  136  139  140  145  149  156  161  163  169  170  172  177  180  183  185  187  188  192  193  199  209  216  220  222  223  225  226  227  231  232  234  236  239  240  241  245  250  270  271  273  275  278  280  281  282  284  285  288  289  293  296  298  300  301  302  307  308  309  310  312  315  323  335  336  337  338  339  341  343  345  346  351  352  359  367  371  372  373  376  377  380  381  382  384  386  392  396  400  401  402  403  406  407  412  415  416  418  420  421  422  424  429  430  432  442  443  447  448  452  453  455  464  465  466  476  478  479  483  484  486  491  494  495  497  501  506  517  519  520  523  524  528  533  536  543  547  549  554  560  561  562  566  567  570  571  573  576  582  583  594  597  599  601  604  613  614  615  622  624  627  630  634  635  641  646  648  649  654  655  657  659  667  669  670  671  673  679  681  682  684  685  686  687  689  697  698  699  701  702  704  708  712  714  715  719  721  723  724  727  733  734  736  739  741  744  748  751  752  758  759  760  763  764  765  767  768  769  770  772  773  783  785  789  792  796  800  805  807  808  809  813  814  815  816  818  823  830  832  833  834  840  842  843  844  849  853  855  858  859  861  862  864  874  880  889  890  898  900  901  903  911  915  922  926  927  937  945  947  953  956  961  966  967  968  970  972  973  976  980  981  984  986  989  990  994  995  996  998 1000 1001 1002 1003 1004 1006 1008 1014 1016 1017 1018 1021 1023 1024 1025 1037 1040 1042 1045 1047 1051 1053 1054 1055 1058 1067 1070 1074 1078 1079 1080 1081 1082 1092 1096 1099 1101 1103 1104 1105 1106 1114 1116 1123 1125 1127 1129 1130 1139 1141 1143 1152 1154 1160 1163 1164 1165 1166 1167 1171 1172 1175 1178 1180 1183 1185 1186 1187 1194 1198 1199 1204 1209 1210 1214 1215 1218 1224 1238 1240 1245 1250 1253 1263 1264 1268 1272 1274 1275 1277 1281 1282 1285 1288 1294 1295 1297 1298 1299 1303 1304 1305 1308 1309 1311 1312 1313 1314 1315 1316 1319 1320 1323 1325 1326 1327 1329 1332 1333 1336 1337 1339 1340 1341 1342 1343 1347 1349 1351 16:    21   26   27   31   35   37   41   57   58   59   69   70   76   77   86   98  101  102  108  110  111  113  116  122  123  125  133  136  139  140  145  149  156  161  163  169  170  172  177  180  183  185  187  188  192  193  199  209  216  220  222  223  225  226  227  231  232  234  236  239  240  241  245  250  270  271  273  275  278  280  281  282  284  285  288  289  293  296  298  300  301  302  307  308  309  310  312  315  323  335  336  337  338  339  341  343  345  346  351  352  359  367  371  372  373  376  377  380  381  382  384  386  392  396  400  401  402  403  406  407  412  415  416  418  420  421  422  424  429  430  432  442  443  447  448  452  453  455  464  465  466  476  478  479  483  484  486  491  494  495  497  501  506  517  519  520  523  524  528  533  536  543  547  549  554  560  561  562  566  567  570  571  573  576  582  583  594  597  599  601  604  613  614  615  622  624  627  630  634  635  641  646  648  649  654  655  657  659  667  669  670  671  673  679  681  682  684  685  686  687  689  697  698  699  701  702  704  708  712  714  715  719  721  723  724  727  733  734  736  739  741  744  748  751  752  758  759  760  763  764  765  767  768  769  770  772  773  783  785  789  792  796  800  805  807  808  809  813  814  815  816  818  823  830  832  833  834  840  842  843  844  849  853  855  858  859  861  862  864  874  880  889  890  898  900  901  903  911  915  922  926  927  937  945  947  953  956  961  966  967  968  970  972  973  976  980  981  984  986  989  990  994  995  996  998 1000 1001 1002 1003 1004 1006 1008 1014 1016 1017 1018 1021 1023 1024 1025 1037 1040 1042 1045 1047 1051 1053 1054 1055 1058 1067 1070 1074 1078 1079 1080 1081 1082 1092 1096 1099 1101 1103 1104 1105 1106 1114 1116 1123 1125 1127 1129 1130 1139 1141 1143 1152 1154 1160 1163 1164 1165 1166 1167 1171 1172 1175 1178 1180 1183 1185 1186 1187 1194 1198 1199 1204 1209 1210 1214 1215 1218 1224 1238 1240 1245 1250 1253 1263 1264 1268 1272 1274 1275 1277 1281 1282 1285 1288 1294 1295 1297 1298 1299 1303 1304 1305 1308 1309 1311 1312 1313 1314 1315 1316 1319 1320 1323 1325 1326 1327 1329 1332 1333 1336 1337 1339 1340 1341 1342 1343 1347 1349 1351 17:   179  207  514  586  591  902 1200 1286 18:   179  207  514  586  591  902 1200 1286 19:    22   29  141  592 20:    25   39   67  105  134  205  304  334  350  388  405  451  454  512  544  676  680  713  737  740  777  797  819  885  912  938  941  978 1038 1062 1064 1089 1121 1140 1142 1191 1290 1344

Recombinant DNA constructs are prepared using the DNA encoding each of the identified homologs and the constructs are used to prepare multiple events of transgenic corn, soybean, canola and cotton plants as illustrated in Examples 2-5. Plants are regenerated from the transformed plant cells and used to produce progeny plants and seed that are screened for enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil. From each group of multiple events of transgenic plants with a specific recombinant DNA for a homolog the event that produces the greatest enhancement in yield, water use efficiency, nitrogen use efficiency, enhanced cold tolerance, enhanced seed protein and enhanced seed oil is identified and progeny seed is selected for commercial development.

Example 7 Consensus Sequence

This example illustrates the identification of consensus amino acid sequence for the proteins and homologs encoded by DNA that is used to prepare the transgenic seed and plants of this invention having enhanced agronomic traits.

ClustalW program was selected for multiple sequence alignments of the amino acid sequence of SEQ ID NO: 17-19 and their homologs. Three major factors affecting the sequence alignments dramatically are (1) protein weight matrices; (2) gap open penalty; (3) gap extension penalty. Protein weight matrices available for ClustalW program include Blosum, Pam and Gonnet series. Those parameters with gap open penalty and gap extension penalty were extensively tested. On the basis of the test results, Blosum weight matrix, gap open penalty of 10 and gap extension penalty of 1 were chosen for multiple sequence alignment.

The consensus amino acid sequence can be used to identify DNA corresponding to the full scope of this invention that is useful in providing transgenic plants, for example corn and soybean plants with enhanced agronomic traits, for example improved nitrogen use efficiency, improved yield, improved water use efficiency and/or improved growth under cold stress, due to the expression in the plants of DNA suppressing a protein with amino acid sequence identical to the consensus amino acid sequence.

The SEQ ID NOs for the identified consensus sequences are reported in table 8 below and the full consensus sequences are provided in the attached sequence listing.

TABLE 8 Gene ID PEP SEQ ID NO Consensus SEQ ID NO Mnom000090 17 1356 Mnom000091 18 1357 Mnom000092 19 1358

Example 9 Identification of Amino Acid Domain by Pfam Analysis

This example illustrates the identification of domain and domain module by Pfam analysis.

The amino acid sequence of the expressed proteins that are shown to be associated with an enhanced trait were analyzed for Pfam protein family against the current Pfam collection of multiple sequence alignments and hidden Markov models using the HMMER software in the appended computer listing. The Pfam protein domains and modules for the proteins of SEQ ID NO: 11 through 16 and 20 are shown in Tables 9, 10 and 11. The Hidden Markov model databases for the identified patent families are also in the appended computer listing allowing identification of other homologous proteins and their cognate encoding DNA to enable the full breadth of the invention for a person of ordinary skill in the art. Certain proteins are identified by a single Pfam domain and others by multiple Pfam domains.

TABLE 9 Pfam annotation PEP SEQ ID NO Gene ID Pfam domain name Begin Stop Score E-value 11 Mnom000034 Cu-oxidase_3 30 146 237 4.10E−68 11 Mnom000034 Cu-oxidase 156 310 158.7 1.50E−44 11 Mnom000034 Cu-oxidase_2 409 532 169.9 6.30E−48 12 Mnom000037 Flavodoxin_1 7 160 197.3 3.70E−56 13 Mnom000048 Glyco_transf_20 3 470 933.9 6.70E−278 13 Mnom000048 Trehalose_PPase 504 748 256.4 6.20E−74 14 Mnom000049 Glyco_transf_20 76 578 841.5 4.40E−250 14 Mnom000049 Trehalose_PPase 627 862 339.1 7.70E−99 15 Mnom000067 Aminotran_1_2 60 441 33.4 8.00E−09 16 Mnom000068 Aminotran_1_2 60 441 33.4 8.00E−09 20 Mnom000095 B3 312 417 118.5 2.00E−32 20 Mnom000095 Auxin_resp 439 524 160.7 3.80E−45 20 Mnom000095 AUX_IAA 681 826 −74.9 0.00043

TABLE 10 Pfam module annotation PEP SEQ ID NO Gene ID Pfam domain module Position 11 Mnom000034 Cu-oxidase_3::Cu- 30-146::156-310::409-532 oxidase::Cu-oxidase_2 12 Mnom000037 Flavodoxin_1 7-160 13 Mnom000048 Glyco_transf_20::Trehalose_PPase 3-470::504-748 14 Mnom000049 Glyco_transf_20::Trehalose_PPase 76-578::627-862 15 Mnom000067 Aminotran_1_2 60-441 16 Mnom000068 Aminotran_1_2 60-441 20 Mnom000095 B3::Auxin_resp::AUX_IAA 312-417::439-524::681-826

TABLE 11 Description of Pfam domains Accession Gathering Pfam domain name number cutoff Domain description AUX_IAA PF02309.8 −83.0000; AUX/IAA family Aminotran_1_2 PF00155.13 −57.5000; Aminotransferase class I and II Auxin_resp PF06507.5 25.0000; Auxin response factor B3 PF02362.13 26.5000; B3 DNA binding domain Cu-oxidase PF00394.14 −18.9000; Multicopper oxidase Cu-oxidase_2 PF07731.6 −5.8000; Multicopper oxidase Cu-oxidase_3 PF07732.7 10.0000; Multicopper oxidase Flavodoxin_1 PF00258.17 6.3000; Flavodoxin Glyco_transf_20 PF00982.13 −243.6000; Glycosyltransferase family 20 Trehalose_PPase PF02358.8 −49.4000; Trehalose-phosphatase 

1. A transgenic plant cell having recombinant DNA in its chromosomal DNA wherein said recombinant DNA comprises (a) a promoter that is functional in a plant cell and that is operably linked to a polynucleotide that encodes a protein having an amino acid sequence having at least 95% identity over at least 95% of the length of SEQ ID NO:13 and (b) a promoter that is functional in a plant cell and that is operably linked to a polynucleotide that encodes a protein having an amino acid sequence having at least 95% identity over at least 95% of the length of SEQ ID NO:14.
 2. A recombinant DNA construct comprising a promoter that is functional in a plant cell and that is operably linked to a polynucleotide that: (a) encodes a protein having an amino acid sequence having at least 95% identity over at least 95% of the length of a reference sequence selected from the group consisting of SEQ ID NO: 12-16, and 20, when said amino acid sequence is aligned to said reference sequence; or (b) is transcribed into an RNA molecule that suppresses the level of an endogenous protein that has an amino acid sequence that is at least 95% identical over at least 95% of the length of a reference sequence of SEQ ID NO: 11, and 17-19, when said amino acid sequence is aligned to said reference sequence; wherein said construct is stably integrated into plant chromosomal DNA.
 3. A transgenic plant cell comprising the recombinant DNA construct of claim 2 wherein said plant cell is in a plant selected by screening a population of transgenic plants that have been transformed with said construct for an enhanced trait as compared to control plants; and wherein said enhanced trait is enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein or enhanced seed oil.
 4. A mixture comprising plant cells of claim 3 and an antibody to a protein produced in said cells wherein said protein has an amino acid sequence that has at least 95% identity over at least 95% of the length of a reference sequence selected from the group consisting of SEQ ID NO: 11-20 when said amino acid sequence is aligned to said reference sequence.
 5. The plant cell of claim 3 further comprising DNA expressing a protein that provides tolerance from exposure to an herbicide comprising an agent applied at levels that are lethal to a wild type of said plant cell.
 6. The plant cell of claim 5 wherein the agent of said herbicide is a glyphosate, dicamba, or glufosinate compound.
 7. A transgenic plant comprising a plurality of plant cells of claim
 3. 8. The transgenic plant of claim 7 which is homozygous for said recombinant DNA.
 9. A transgenic seed comprising a plurality of plant cells of claim
 3. 10. The transgenic seed of claim 9 from a corn, soybean, cotton, canola, alfalfa, wheat, rice, sugarcane, or sugar beet plant.
 11. Grain comprising transgenic seed identifiable by the recombinant DNA construct of claim
 2. 12. Seed meal produced from transgenic seed identifiable by the recombinant DNA construct of claim
 2. 13. A transgenic pollen grain comprising a haploid derivative of a plant cell nucleus having a chromosome comprising the recombinant DNA construct of claim
 2. 14. A method for manufacturing non-natural, transgenic seed that can be used to produce a crop of transgenic plants with an enhanced trait resulting from expression of the stably-integrated, recombinant DNA construct of claim 2, said method comprising: (a) screening a population of plants for said enhanced trait and said recombinant DNA, wherein individual plants in said population exhibit said trait at a level less than, essentially the same as or greater than the level that said trait is exhibited in control plants which do not contain said recombinant DNA, wherein said enhanced trait is selected from the group of enhanced traits consisting of enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil; (b) selecting from said population one or more plants that exhibit said trait at a level greater than the level that said trait is exhibited in control plants, and (c) collecting seed from selected plant from step b.
 15. The method of claim 14 wherein said method for manufacturing said transgenic seed further comprises: (a) verifying that said recombinant DNA is stably integrated in said selected plants, and (b) analyzing tissue of said selected plant to determine the expression or suppression of a protein having the function of a protein having an amino acid sequence selected from the group consisting of one of SEQ ID NOs:11-20.
 16. The method of claim 15 wherein said seed is corn, soybean, cotton, canola, alfalfa, wheat, rice, sugarcane, or sugar beet seed.
 17. A method of producing hybrid corn seed comprising: (a) acquiring hybrid corn seed from an herbicide tolerant corn plant which also has the stably-integrated, recombinant DNA construct of claim 2; (b) producing corn plants from said hybrid corn seed, wherein a fraction of the plants produced from said hybrid corn seed is homozygous for said recombinant DNA, a fraction of the plants produced from said hybrid corn seed is hemizygous for said recombinant DNA, and a fraction of the plants produced from said hybrid corn seed has none of said recombinant DNA; (c) selecting corn plants which are homozygous and hemizygous for said recombinant DNA by treating with an herbicide; (d) collecting seed from herbicide-treated-surviving corn plants and planting said seed to produce further progeny corn plants; (e) repeating steps (c) and (d) at least once to produce an inbred corn line; and (f) crossing said inbred corn line with a second corn line to produce hybrid seed. 